Composition for inhibiting growth of bacterium having menaquinone synthesis pathway through futalosine or futalosine derivative

ABSTRACT

The present invention provides a composition for inhibiting proliferation of a bacterium provided with a menaquinone synthesis route via futalosine or a futalosine derivative, which contains a fatty acid having 18 carbon atoms and a hydroxyl group at the 10-position as an active ingredient. Furthermore, the present invention provides a composition for preventing or treating a disease caused by a bacterium provided with a menaquinone synthesis route via futalosine or a futalosine derivative.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage of International ApplicationNo. PCT/JP2018/006804, filed Feb. 23, 2018, which claims the benefit ofJapanese Patent Application No. 2017-053056, filed Mar. 17, 2017, theentire contents of each of which are fully incorporated herein byreference.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

A Sequence Listing, which is a part of the present disclosure, issubmitted concurrently with the specification as a text file. The nameof the text file containing the Sequence Listing is “54774Seglisting.txt.” The Sequence Listing was created on Sep. 16, 2019, andis 2,177 bytes in size. The subject matter of the Sequence Listing isincorporated herein in its entirety by reference.

TECHNICAL FIELD

The present invention relates to a composition containing a fatty acidhaving 18 carbon atoms and a hydroxyl group at the 10-position as anactive ingredient for suppressing proliferation of a bacterium providedwith a menaquinone synthesis route via futalosine or a futalosinederivative (preferably, Helicobacter bacterium or Campylobacterbacterium). Furthermore, the present invention relates to a compositionfor preventing or treating a disease caused by Helicobacter bacterium ora composition for preventing or treating a disease caused byCampylobacter bacterium. In addition, the present invention also relatesto the composition as a food, a pharmaceutical product, a feed or thelike.

BACKGROUND ART

At present, about 50% of the population is considered to haveHelicobacter bacterium in the gastrointestinal tract. Helicobacterbacterium carriers may not have subjective symptoms but it is known thatgastritis, stomach pain or stomach cancer is developed. Since the routeof infection with Helicobacter bacterium is oral or fecal route, theinfection rate of Helicobacter bacteria is high in the developingcountries where hygiene state is poor.

When infected with Helicobacter pylori, which is one kind ofHelicobacter bacteria, the infection is detected by a urease test(13C-UREA BREATH TEST), and is treated by eradication. As theeradication method, a three agent combination therapy using a protonpump inhibitor that suppresses secretion of gastric acid and antibiotics(penicillin-based and macrolide-based) has been established. Theeradication rate is said to be about 90% and Helicobacter pylori thatcould not be eradicated may proliferate again. In addition, thiseradication method has been reported to cause side effects such asdiarrhea, gustation disorder, allergy, emergence of multiple drugresistant bacteria and the like. The side effects are considered to bemainly caused by changes in the intestinal bacterial flora due to theadministration of a large amount of antibiotics having broad spectrum.

Among the Helicobacter bacteria that infect the human stomach,Helicobacter suis is difficult to diagnose since it is often negativefor the urease test even if infection is present and is a hardlyculturable bacterium. Thus, an eradication method has not beenestablished. Furthermore, about 60% of the patients who developedgastric MALT lymphoma, which is one type of stomach cancer, shownegative Helicobacter pylori test results but are known to be infectedwith bacteria such as Helicobacter suis and the like included inHelicobacter heilmannii sensu lato.

Poultry infected with Campylobacter bacteria is a major cause of foodpoisoning in human. In particular, the carrying rate of the distributingchicken is said to be high. While the possibility of contamination withCampylobacter bacteria in poultry houses and chicken processing plantsis mentioned, the exact source of contamination is unknown even now.Fever and gastrointestinal inflammation are the main symptoms of humanCampylobacter infection, and the complications thereof includeGuillain-Barre syndrome which is a neurological disorder.

As a probiotic bacterium that inhibits proliferation of Helicobacterbacteria, Lactobacillus gasseri has been reported (patent document 1,non-patent document 1). It is well known that menaquinone (vitamin K2)is an essential component of the electron transport system of bacteria.It is suggested that Helicobacter bacteria and Campylobacter bacteriaare provided with a menaquinone synthesis route via futalosine or afutalosine derivative (non-patent document 2), and this synthesis routeis different from the menaquinone synthesis route that Lactobacillus,Escherichia coli and the like have. It has heretofore been reported thatstraight chain unsaturated fatty acid (patent document 2, non-patentdocument 3, non-patent document 4) and branched saturated fatty acid(non-patent document 5), which are considered to target menaquinonesynthesis route via futalosine or a futalosine derivative, have aneffect of inhibiting proliferation of Helicobacter pylori. Furthermore,foods and drinks for eradicating Helicobacter pylori containing freehydroxy fatty acid as an active ingredient have been reported. (patentdocument 3). In addition, as a compound inhibiting proliferation ofCampylobacter bacterium, caprylic acid, which is a middle chain fattyacid, is known (non-patent document 6).

As mentioned above, since the synthesis route of menaquinone variesdepending on the bacterium, a compound capable of more strongly blockingonly the menaquinone synthesis route possessed by pathogenicmicroorganisms including Helicobacter bacterium and Campylobacterbacterium can inhibit proliferation of pathogenic microorganisms withoutbreaking intestinal bacterial flora. Therefore, a compound capable ofmore efficiently inhibiting menaquinone synthesis route via futalosineor a futalosine derivative has been demanded.

DOCUMENT LIST Patent Documents

-   patent document 1: JP-A-2015-117220-   patent document 2: US2011/0064789A1-   patent document 3: JP-A-2002-85012

Non-Patent Document

-   non-patent document 1: H. Matsui, et al., Mouse Models for Assessing    the Protective Efficacy of Lactobacillus gasseri SBT2055 against    Helicobacter suis Infection Associated with the Development of    Gastric Mucosa-Associated Lymphoid Tissue Lymphoma, Helicobacter,    2015; 20: 291-298-   non-patent document 2: T. Hiratsuka, et al., An Alternative    Menaquinone Biosynthetic Pathway Operating in Microorganisms,    Science, 2008 Sep. 19; 321(5896): 1670-3-   non-patent document 3: T. Yamamoto, et al., Narrow-spectrum    inhibitors targeting an alternative menaquinone biosynthetic pathway    of Helicobacter pylori, J. Infect. Chemother., 2016 September;    22(9):587-92-   non-patent document 4: L. Thompson, et al., Inhibitory effect of    polyunsaturated fatty acids on the growth of Helicobacter pylori: a    possible explanation of the effect of diet on peptic ulceration,    Gut, 1994; 35: 1557-1561-   non-patent document 5: R. Tanaka, et al., Branched fatty acids    inhibit the biosynthesis of menaquinone in Helicobacter pylori, J.    Antibiot., 2011; 64: 151-153-   non-patent document 6: F. Solis de los Santos, et al., Therapeutic    Supplementation of Caprylic Acid in Feed Reduces Campylobacter    jejuni Colonization in Broiler Chicks, Appl. Environ. Microbiol.,    2008 July; 74 (14): 4564-6

SUMMARY OF THE INVENTION Problems to be Solved by the Invention

It is an object of the present invention to find a compound thatinhibits menaquinone synthesis route via futalosine or a futalosinederivative more efficiently from known compounds that inhibit the route,and provide a composition for inhibiting of a bacterium provided withmenaquinone synthesis route via futalosine or a futalosine derivative,which contains the compound as an active ingredient, and further, acomposition for preventing or treating a disease caused by a bacteriumprovided with a menaquinone synthesis route via futalosine or afutalosine derivative.

Means of Solving the Problems

In view of the above-mentioned problems, the present inventors haveconducted intensive studies and found that a fatty acid having 18 carbonatoms and a hydroxyl group at the 10-position has an inhibitory actionon the proliferation of Helicobacter pylori and Helicobacter suis, asuppressive action on an increase in the number of Ki-67 positive cells,a suppressive action on an increase in the CD19 expression level andCD20 expression level, and a suppressive action on the onset ofpathology of gastric MALT lymphoma, which are physiological functionsnot conventionally known. Also, the present inventors have found that afatty acid having 18 carbon atoms and a hydroxyl group at the10-position has an inhibitory action on proliferation of Campylobacterjejuni and Campylobacter coli.

Based on the above findings, the present invention has been completed.

That is, the present invention is as follows.

[1] A composition for inhibiting proliferation of a bacterium providedwith a menaquinone synthesis route via futalosine or a futalosinederivative, comprising a fatty acid having 18 carbon atoms and ahydroxyl group at the 10-position.

[2] The composition of [1] wherein the fatty acid has a cis double bondat at least the 12-position.

[3] The composition of [2] wherein the fatty acid is at least oneselected from the group consisting of 10-hydroxy-cis-12-octadecenoicacid, 10-hydroxy-cis-12,cis-15-octadecadienoic acid and10-hydroxy-cis-6,cis-12-octadecadienoic acid.

[4] The composition of [3] wherein the fatty acid is10-hydroxy-cis-12-octadecenoic acid.

[5] The composition of any one of [1] to [4] wherein the bacterium isHelicobacter bacterium.

[6] The composition of [5] wherein the Helicobacter bacterium isselected from the group consisting of Helicobacter pylori andHelicobacter suis.

[7] The composition of [5] or [6] for use in preventing or treating adisease selected from the group consisting of acute gastritis, chronicgastritis, nodular gastritis, gastric ulcer, duodenal ulcer, stomachcancer, stomach MALT lymphoma, diffuse large B-cell lymphoma, idiopathicthrombocytopenic purpura, childhood hypoferric anemia, chronic urticariaand Parkinson's disease.[8] The composition of any one of [1] to [4] wherein the bacterium isCampylobacter bacterium.[9] The composition of [8] wherein the Campylobacter bacterium isselected from the group consisting of Campylobacter jejuni andCampylobacter coli.[10] The composition of [8] or [9] for use in preventing or treatingCampylobacter food poisoning or Guillain-Barre syndrome.[11] The composition of any one of [1] to [10] which is a food or a foodadditive.[12] The composition of any one of [1] to [10] which is a pharmaceuticalproduct.[13] The composition of any one of [1] to [10] which is a feed or a feedadditive.[14] A method for inhibiting proliferation of a bacterium provided witha menaquinone synthesis route via futalosine or a futalosine derivative,comprising administering a fatty acid having 18 carbon atoms and ahydroxyl group at the 10-position to a subject.[15] A method for preventing or treating a disease selected from thegroup consisting of acute gastritis, chronic gastritis, nodulargastritis, gastric ulcer, duodenal ulcer, stomach cancer, stomach MALTlymphoma, diffuse large B-cell lymphoma, idiopathic thrombocytopenicpurpura, childhood hypoferric anemia, chronic urticaria and Parkinson'sdisease, comprising administering a fatty acid having 18 carbon atomsand a hydroxyl group at the 10-position to a subject.[16] A method for preventing or treating Campylobacter food poisoning orGuillain-Barre syndrome, comprising administering a fatty acid having 18carbon atoms and a hydroxyl group at the 10-position to a subject.[17] A fatty acid having 18 carbon atoms and a hydroxyl group at the10-position for use in preventing or treating a disease selected fromthe group consisting of acute gastritis, chronic gastritis, nodulargastritis, gastric ulcer, duodenal ulcer, stomach cancer, stomach MALTlymphoma, diffuse large B-cell lymphoma, idiopathic thrombocytopenicpurpura, childhood hypoferric anemia, chronic urticaria and Parkinson'sdisease.[18] A fatty acid having 18 carbon atoms and a hydroxyl group at the10-position for use in preventing or treating Campylobacter foodpoisoning or Guillain-Barre syndrome.[19] Use of a fatty acid having 18 carbon atoms and a hydroxyl group atthe 10-position in the production of a proliferation inhibitor of abacterium provided with a menaquinone synthesis route via futalosine ora futalosine derivative.[20] Use of a fatty acid having 18 carbon atoms and a hydroxyl group atthe 10-position in the production of an agent for preventing or treatinga disease selected from the group consisting of acute gastritis, chronicgastritis, nodular gastritis, gastric ulcer, duodenal ulcer, stomachcancer, stomach MALT lymphoma, diffuse large B-cell lymphoma, idiopathicthrombocytopenic purpura, childhood hypoferric anemia, chronic urticariaand Parkinson's disease.[21] Use of a fatty acid having 18 carbon atoms and a hydroxyl group atthe 10-position in the production of an agent for preventing or treatingCampylobacter food poisoning or Guillain-Barre syndrome.

Effect of the Invention

The present invention provides a composition containing a fatty acidhaving 18 carbon atoms and a hydroxyl group at the 10-position as anactive ingredient for suppressing proliferation of a bacterium providedwith a menaquinone synthesis route via futalosine or a futalosinederivative, and further, a composition for preventing or treating adisease caused by a bacterium provided with a menaquinone synthesisroute via futalosine or a futalosine derivative. The compositions can beused in various fields such as pharmaceutical product, food, feed andthe like, and thus the present invention is industrially extremelyuseful.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1-1 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori SS1 strain.

FIG. 1-2 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori TN2GF4 strain.

FIG. 1-3 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori ATCC43579 strain.

FIG. 1-4 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori NCTC11637 strain.

FIG. 1-5 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori TY281 strain.

FIG. 1-6 shows a proliferation suppressive effect (in vitro) of HYA onHelicobacter pylori TY1345 strain.

FIG. 2-1 shows a proliferation suppressive effect (in vitro) of lowconcentration HYA on Helicobacter pylori SS1 strain.

FIG. 2-2 shows a proliferation suppressive effect (in vitro) of lowconcentration HYA on Helicobacter pylori TN2GF4 strain.

FIG. 2-3 shows a proliferation suppressive effect (in vitro) of lowconcentration HYA on Helicobacter pylori TK1029 strain.

FIG. 2-4 shows a proliferation suppressive effect (in vitro) of lowconcentration HYA on Helicobacter pylori RC-1 strain.

FIG. 3-1 shows a proliferation suppressive effect (in vivo) of fattyacid on Helicobacter pylori SS1 strain.

FIG. 3-2 shows a proliferation suppressive effect (in vivo) of fattyacid on Helicobacter pylori TN2GF4 strain.

FIG. 4-1 shows a proliferation suppressive effect (in vivo) of HYA onHelicobacter pylori SS1 strain.

FIG. 4-2 shows a proliferation suppressive effect (in vivo) of HYA onHelicobacter pylori TN2GF4 strain.

FIG. 4-3 shows a proliferation suppressive effect (in vivo) of HYA onHelicobacter suis TKY strain or SNTW101 strain.

FIG. 5-1 shows a proliferation suppressive effect (in vivo) of HYA onHelicobacter suis TKY strain.

FIG. 5-2 shows a suppressive effect (in vivo) of HYA on an increase inthe number of Ki-67 positive cells in a stomach tissue of Helicobactersuis TKY strain-infected mouse.

FIG. 5-3 shows a suppressive effect (in vivo) of HYA on an increase inCD20 expression in a stomach tissue of Helicobacter suis TKYstrain-infected mouse.

FIG. 5-4 shows a suppressive effect (in vivo) of HYA on an increase inCD19 expression in a stomach tissue of Helicobacter suis TKYstrain-infected mouse.

FIG. 6-1 shows a proliferation suppressive effect (in vitro) of HYA onCampylobacter jejuni ATCC33560 strain.

FIG. 6-2 shows a proliferation suppressive effect (in vitro) of HYA onCampylobacter coli ATCC33559 strain.

FIG. 6-3 shows a proliferation suppressive effect (in vitro) of HYA onCampylobacter jejuni ATCC33560 strain.

FIG. 6-4 shows a proliferation suppressive effect (in vitro) of HYA onCampylobacter coli ATCC33559 strain.

DESCRIPTION OF EMBODIMENTS

The present invention provides a composition for inhibitingproliferation of a bacterium provided with a menaquinone synthesis routevia futalosine or a futalosine derivative, which contains a fatty acidhaving 18 carbon atoms and a hydroxyl group at the 10-position(hereinafter to be also referred to as the composition of the presentinvention).

The composition of the present invention contains a fatty acid having 18carbon atoms and a hydroxyl group at the 10-position (hereinafter to bealso referred to as the hydroxylated fatty acid in the presentinvention). The hydroxylated fatty acid in the present invention may bea saturated fatty acid or an unsaturated fatty acid. When it is anunsaturated fatty acid, an unsaturated fatty acid having at least onedouble bond selected from the group consisting of a cis double bond atthe 6-position, a cis double bond at the 12-position, a cis double bondat the 15-position and a trans double bond at the 11-position ispreferable, an unsaturated fatty acid having a cis double bond at atleast the 12-position is more preferable.

More specifically, examples of the hydroxylated fatty acid in thepresent invention include 10-hydroxy-cis-12-octadecenoic acid(hereinafter to be also referred to as HYA),10-hydroxy-cis-12,cis-15-octadecadienoic acid (hereinafter to be alsoreferred to as αHYA), 10-hydroxy-cis-6,cis-12-octadecadienoic acid(hereinafter to be also referred to as γHYA),10-hydroxy-cis-6,cis-12,cis-15-octadecatrienoic acid (hereinafter to bealso referred to as sHYA), 10,12-dihydroxyoctadecanoic acid (hereinafterto be also referred to as rHYA), 10-hydroxy-trans-11-octadecenoic acid(hereinafter to be also referred to as HYC),10-hydroxy-trans-11,cis-15-octadecadienoic acid (hereinafter to be alsoreferred to as αHYC), 10-hydroxy-cis-6,trans-11-octadecadienoic acid(hereinafter to be also referred to as γHYC),10-hydroxy-cis-6,trans-11,cis-15-octadecatrienoic acid (hereinafter tobe also referred to as sHYC) and the like. It is preferably10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid or10-hydroxy-cis-6,cis-12-octadecadienoic acid, further preferably10-hydroxy-cis-12-octadecenoic acid.

The hydroxylated fatty acid in the present invention can be prepared bya known means and, for example, a production method is also described inWO 2013/168310. In addition, 10-hydroxy-cis-12-octadecenoic acid can beprepared by reference to Biochemical and Biophysical ResearchCommunications, 416(2011), p. 188-193, and the like.

The hydroxylated fatty acid in the present invention has a proliferationinhibitory effect on a bacterium provided with a menaquinone synthesisroute via futalosine or a futalosine derivative (hereinafter to be alsoreferred to as futalosine synthesis bacterium). In the presentinvention, the futalosine synthesis bacterium is a bacterium providedwith, in a metabolism pathway for biosynthesizing menaquinone, ametabolism pathway for biosynthesizing menaquinone from chorismic acidvia futalosine or a futalosine derivative (hereinafter to be alsoreferred to as futalosine pathway). As used herein, the futalosinederivative is, for example, aminodeoxyfutalosine, dehypoxanthinylfutalosine, cyclic dehypoxanthine futalosine or 1,4-dihydroxy-6-naphtoicacid. In the futalosine pathway, futalosine is synthesized fromchorismic acid and inosine. Aminodeoxyfutalosine is synthesized fromchorismic acid and adenosine. Futalosine or aminodeoxyfutalosine ismetabolized into dehypoxanthinyl futalosine, dehypoxanthinyl futalosineis metabolized into cyclic dehypoxanthine futalosine, cyclicdehypoxanthine futalosine is metabolized into 1,4-dihydroxy-6-naphtoicacid, and finally, 1,4-dihydroxy-6-naphthoic acid is metabolized intomenaquinone.

Menaquinone is an essential component in the electron transport systemof bacteria, and two kinds of routes are known as synthesis routes inthe bacteria. One is a route for synthesizing menaquinone from chorismicacid via succinylbenzoic acid (hereinafter to be also referred to assuccinylbenzoic acid route), and it is provided in Escherichia coli,Lactobacillus, bifidobacteria, Enterococcus, Salmonella, Shigella,Listeria, Yersinia, Bacillus subtilis and the like. The other is afutalosine pathway clarified by genetic analysis in recent years. As abacterium having the both pathways of succinylbenzoic acid pathway andfutalosine pathway, Stackebrandtia nassauensis DSM 44728 which is onespecies of actinomycetes is known to date. Except this bacterium, abacterium provided with a metabolism pathway for biosynthesizingmenaquinone is only provided with any one route of the above-mentionedroutes. Therefore, the hydroxylated fatty acid in the present inventioncapable of inhibiting the futalosine pathway can specifically inhibitproliferation of futalosine synthesis bacterium. In the presentinvention, the futalosine synthesis bacterium is not particularlylimited as long as it is provided with a futalosine pathway, and abacterium not provided with a succinylbenzoic acid pathway ispreferable. Examples of the futalosine synthesis bacterium includeHelicobacter bacterium, Campylobacter bacterium, Chlamydia bacterium,Thermus bacterium, Wolinella bacterium, Streptomyces bacterium,Acidothermus bacterium, Kitasatospora bacterium, Bacillus bacterium andthe like. In the present invention, examples of the Helicobacterbacterium include Helicobacter pylori, Helicobacter heilmannii sensulato (including Helicobacter suis, Helicobacter felis, Helicobactersalomonis, Helicobacter bizzozeronii, Helicobacter baculiformis,Helicobacter cynogastricus, Helicobacter heilmannii sensu stricto),Helicobacter anseris, Helicobacter acinonychis, Helicobacter bilis,Helicobacter brantae, Helicobacter canadensis, Helicobacter canis,Helicobacter cholecystus, Helicobacter cinaedi, Helicobacter hepaticus,Helicobacter muridarum, Helicobacter mustelae, Helicobacter pametensis,Helicobacter rodentium, Helicobacter trogontum and the like, preferably,Helicobacter pylori and Helicobacter suis. In the present invention,examples of the Campylobacter bacterium include Campylobacter coli,Campylobacter concisus, Campylobacter fetus, Campylobacter jejuni,Campylobacter sputorum, Campylobacter mucosalis, Campylobacter rectusand the like, preferably, Campylobacter jejuni and Campylobacter coli.In the present invention, examples of the Chlamydia bacterium includeChlamydia muridarum, Chlamydia suis, Chlamydia trachomatis and the like.In the present invention, examples of the Thermus bacterium includeThermus antranikianii, Thermus aquaticus, Thermus igniterrae, Thermusthermophilus and the like. In the present invention, examples of theWolinella bacterium include Wolinella curva, Wolinella succinogenes,Wolinella recta and the like. In the present invention, examples of theStreptomyces bacterium include Streptomyces avermitilis, Streptomycescoelicolor, Streptomyces scabies, Streptomyces lividans and the like. Inthe present invention, examples of the Acidothermus bacterium includeAcidothermus cellulolyticus and the like. In the present invention,examples of the Kitasatospora bacterium include Kitasatospora setae andthe like. In the present invention, examples of the Bacillus bacteriuminclude Bacillus cereus, Bacillus subtilis, Bacillus thuringiensis andthe like.

The hydroxylated fatty acid in the present invention can inhibitproliferation of futalosine synthesis bacterium, and thus can be used,when the futalosine synthesis bacterium is a bacterium that infectshuman or animals other than human and cause a disease (hereinafter to bealso referred to as pathogenic futalosine synthesis bacterium), for theprophylaxis or treatment of the disease. Examples of the pathogenicfutalosine synthesis bacterium include Helicobacter bacterium,Campylobacter bacterium, Chlamydia bacterium, Wolinella bacterium,Bacillus bacterium and the like. When the pathogenic futalosinesynthesis bacterium is Helicobacter bacterium, examples of the diseasethat can be prevented or treated by the composition of the presentinvention include gastric diseases such as acute gastritis, chronicgastritis, nodular gastritis, gastric ulcer, duodenal ulcer, stomachcancer, gastric MALT lymphoma and the like, non-gastric diseases such asdiffuse large B-cell lymphoma, idiopathic thrombocytopenic purpura,childhood hypoferric anemia, chronic urticaria and the like, Parkinson'sdisease and the like, preferably, gastric diseases such as acutegastritis, chronic gastritis, nodular gastritis, gastric ulcer, duodenalulcer, stomach cancer, gastric MALT lymphoma and the like. When thepathogenic futalosine synthesis bacterium is Campylobacter bacterium,examples of the disease that can be prevented or treated by thecomposition of the present invention include Campylobacter foodpoisoning, Guillain-Barre syndrome and the like. When the pathogenicfutalosine synthesis bacterium is Chlamydia bacterium, examples of thedisease that can be prevented or treated by the composition of thepresent invention include Chlamydia infections, trachoma, pneumonia,parrot disease and the like. When the pathogenic futalosine synthesisbacterium is Wolinella bacterium, examples of the disease that can beprevented or treated by the composition of the present invention includeperiodontitis and the like. When the pathogenic futalosine synthesisbacterium is Bacillus bacterium, examples of the disease that can beprevented or treated by the composition of the present invention includefood poisoning, bacteremia, pneumonia, endocarditis, ocular infections,opportunistic infection and the like.

The composition of the present invention can be used as, for example,pharmaceutical product, food, feed and the like, or by blending withthem.

When the composition of the present invention is used as apharmaceutical product, the dosage form of the pharmaceutical productincludes dispersion, granule, pill, soft capsule, hard capsule, tablet,chewable tablet, quick-disintegrating tablet, syrup, liquid, suspension,suppository, ointment, cream, gel, adhesive, inhalant, injection and thelike. A preparation thereof is prepared according to a conventionalmethod.

Examples of the additives that can be used for forming preparationsinclude animal and plant oils such as soybean oil, safflower oil, oliveoil, germ oil, sunflower oil, beef tallow, sardine oil and the like,polyvalent alcohols such as polyethylene glycol, propylene glycol,glycerol, sorbitol and the like, surfactants such as sorbitan fatty acidester, sucrose fatty acid ester, glycerin fatty acid ester, polyglycerolfatty acid ester and the like, excipients such as purified water,lactose, starch, crystalline cellulose, D-mannitol, lecithin, gumarabic, sorbitol solution, carbohydrate solution and the like,sweetener, colorant, pH adjuster, flavor and the like. A liquidpreparation may be dissolved or suspended in water or other suitablemedium when in use. Also, tablet and granules may be coated by awell-known method.

For administration in the form of an injection, intravenous,intraperitoneal, intramuscular, subcutaneous, transdermal,intraarticular, intrasynovial, intrathecal, intraperiosteum, sublingual,oral administrations and the like are preferable, and intravenousadministration or intraperitoneal administration is particularlypreferable. The intravenous administration may be any of dripadministration and bolus administration.

When the composition of the present invention is used as a food or foodadditive, the food of the present invention is not particularly limitedas long as it permits oral ingestion, such as solution, suspension,powder, solid formed article and the like. Specific examples includesupplements (dispersion, granule, soft capsule, hard capsule, tablet,chewable tablet, quick-disintegrating tablet, syrup, liquid etc.),drinks (carbonic acid drinks, lactic acid drinks, sport drinks, fruitjuice drinks, vegetable drinks, soymilk drinks, coffee drinks, teadrinks, powder drinks, concentrated drinks, nutrition drinks, alcoholdrinks etc.), confectionery (gummy, jelly, gum, chocolate, cookie,candy, caramel, Japanese confectionery, snack etc.), instant food(instant noodles, retort food, can, microwavable foods, instant soup,miso soups, freeze-dried food etc.), oil, fats and oils food(mayonnaise, dressing, butter, cream, margarine etc.), wheat powderproducts (bread, pasta, noodle, cake mix, bread crumb etc.), seasoning(sauce, tomato processing seasoning, flavor seasoning, cooking mixture,soup etc.), processed meat products (meat ham, sausage and the like).

The above-mentioned foods can contain, where necessary, variousnutrients, various vitamins (vitamin A, vitamin B1, vitamin B2, vitaminB6, vitamin C, vitamin D, vitamin E, vitamin K etc.), various minerals(magnesium, zinc, iron, sodium, potassium, selenium etc.), dietaryfiber, dispersing agent, stabilizer such as emulsifier and the like,sweetener, flavor components (citric acid, malic acid etc.), flavor,royal jelly, propolis, Agaricus and the like.

When the composition of the present invention is used as a feed or feedadditive, the feed of the present invention is, for example, pet food,stock raising or aquaculture feed additive and the like.

As the subject to be administered with or that ingests the compositionof the present invention, human and animals other than human (e.g., dog,cat, mouse, rat, hamster, guinea pig, rabbit, swine, bovine, chicken,parakeet, hill myna, goat, horse, sheep, monkey etc.) can be mentioned.

While the dose or ingestion amount of the composition of the presentinvention varies depending on the subject of administration oringestion, target disease, symptom, administration or ingestion routeand the like, for example, a daily dose or ingestion amount of the fattyacid contained in the composition of the present invention is generally0.02-100 mg/kg body weight, preferably 0.2-50 mg/kg body weight, morepreferably 0.5-20 mg/kg body weight, which can be administered oringested orally or parenterally. Plural divided portions may beadministered or ingested per day. The dose may be increased or decreasedaccording to the symptom.

The present invention is explained in more detail in the following byreferring to Examples. The Examples are mere exemplifications of thepresent invention and do not limit the scope of the present invention inany manner.

EXAMPLES

Strain

The strains used in this Example are as follows. Helicobacter pylori SS1strain is an experimental strain isolated from a patient with a stomachdisease and adapted to infect mice. Helicobacter pylori TN2GF4 strain,NCTC11637 strain, ATCC43579 strain, TK1029 strain, TY281 strain andTY1345 strain are isolated from gastric biopsy specimens of patientswith stomach diseases. Helicobacter pylori RC-1 strain is isolated froma gastric biopsy specimen of a patient with a stomach disease and isclarithromycin resistant. Helicobacter suis TKY strain is isolated fromMacaca fascicularis. Helicobacter suis SNTW101 strain is isolated from apatient who developed nodular gastritis. Campylobacter jejuni ATCC33560strain is a standard strain of Campylobacter jejuni isolated from bovinefeces. Campylobacter coli ATCC33559 strain is a standard strain ofCampylobacter coli isolated from swine feces.

Manufacture or Preparation of Fatty Acids

The fatty acid having 18 carbon atoms and a hydroxyl group or a carbonylgroup at the 10-position (10-hydroxyoctadecanoic acid (hereinafter to bealso referred to as HYB), 10-oxo-cis-12-octadecenoic acid (hereinafterto be also referred to as KetoA) and 10-oxo-trans-11-octadecenoic acid(hereinafter to be also referred to as KetoC)) to be used in thisExample were prepared according to the method of WO 2013/168310. Inaddition, HYA, αHYA, γHYA were prepared by reference to the report inBiochemical and Biophysical Research Communications 416 (2011) p.188-193, and the like. The stearic acid, oleic acid, linoleic acid,ricinoleic acid, DHA and EPA to be used in this Example were purchasedfrom Nacalai Tesque, INC. The ricinoleic acid to be used in this Examplewas purchased from Sigma-Aldrich Co. LLC.

Example 1 Proliferation Inhibitory Effect (In Vitro) on Helicobacterpylori TK1029 Strain by ED₅₀ Measurement

Helicobacter pylori TK1029 strain (clinical isolate) was cultured inBrucella broth containing 10% FCS and 1×10⁶ CFU of bacteria wererespectively added to 3 mL of Brain-Heart Infusion Broth (BHI) media(previously added with free fatty acid (stearic acid, oleic acid,linoleic acid, ricinoleic acid, HYA) at 0, 0.5, 5, 50, 500 μg/mL)containing 2% FCS in a 6-well plate. Shaking culture was performed underconditions of temperature 37° C., humidity 100%, microaerobic (5% O₂,10% CO₂, 85% N₂), the culture medium (0.1 mL) from each well was appliedto NISSUI plate, Helicobacter agar medium 15 hr and 25 hr later, and thenumber of colonies formed after microaerobic culture for 3 days wascounted. This viable count is expressed as a logarithm and, on thelogarithmic graph where the detection limit of viable count is 10², theconcentration at which the number of viable cells in the medium addedwith free fatty acid is half the number of viable cells in the mediumnot added with free fatty acid was taken as the ED₅₀ value, (ED₅₀ valueis a concentration at which the viable count of the medium added withfree fatty acid is {10× (viable count of the medium not added with freefatty acid)^(1/2)}). As a result, HYA already showed ananti-Helicobacter pylori effect at 15 hr after culturing as evidenced byED₅₀ value about 1000-100 times lower than that of fatty acid having 18carbon atoms and not having a hydroxyl group (stearic acid, oleic acid,linoleic acid), and showed an anti-Helicobacter pylori effect asevidenced by ED₅₀ value about 10 times lower than that of ricinoleicacid which is a fatty acid having 18 carbon atoms and a hydroxyl group(Table 1). Even at 25 hr after culturing, HYA showed ananti-Helicobacter pylori effect with ED₅₀ values remarkably lower thanor the same level as those of the above-mentioned fatty acids.

TABLE 1 fatty acid 15 hr later (μg/mL) 25 hr later (μg/mL) stearic acid500 >500 oleic acid 50 >50 linoleic acid 50 50 ricinoleic acid 5 0.5 HYA0.5 0.5

Example 2 Proliferation Inhibitory Effect (In Vitro) of HYA on 20Helicobacter pylori

Helicobacter pylori SS1 strain, TN2GF4 strain, ATCC43579 strain,NCTC11637 strain, TY281 strain, TY1345 strain were each inoculated to a6-well plate containing 3 mL of a liquid medium (Brucella broth addedwith 10% FCS), and fatty acid (200 μM or 1000 μM) and menaquinone (MK-4)100 μg/mL were added. After static culture for 3 days at temperature 37°C., microaerobic conditions (5% O₂, 10% CO₂, 85% N₂), the culture mediumwas applied on a Helicobacter agar plate, cultured for 3 days and theresulting colonies were counted (FIG. 1-1 A: SS1 strain, FIG. 1-2 B:TN2GF4 strain, FIG. 1-3 C: ATCC43579 strain, FIG. 1-4 D: NCTC11637strain, FIG. 1-5 E: TY281 strain, FIG. 1-6 F: TY1345 strain. Item C:control (no addition), Item 1: HYA addition, Item 2: HYA and menaquinoneaddition, Item 3: linoleic acid addition, Item 4: linoleic acid andmenaquinone addition). HYA suppressed proliferation of Helicobacterpylori SS1 strain, TN2GF4 strain, ATCC43579 strain, NCTC11637 strain,TY281 strain, TY1345 strain more than linoleic acid. Addition ofmenaquinone (MK-4) mitigated inhibition of proliferation of Helicobacterpylori by fatty acid.

Example 3 Proliferation Inhibitory Effect (In Vitro) of LowConcentration HYA on Helicobacter pylori

Helicobacter pylori SS1 strain, TN2GF4 strain, TK1029 strain, RC-1strain were each inoculated to a 6-well plate containing 2 mL of aliquid medium (BHI added with 5% FCS), and fatty acid (20 μM) andmenaquinone (MK-4) 100 μg/mL were added. After shaking culture for 24 hrat temperature 37° C., microaerobic conditions (5% O₂, 10% CO₂, 85% N₂),the culture medium was applied on a Helicobacter agar plate, culturedfor 3 days and the resulting colonies were counted (FIG. 2-1 A: SS1strain, FIG. 2-2 B: TN2GF4 strain, FIG. 2-3 C: TK1029 strain, FIG. 2-4D: RC-1 strain. Item 1: control (menaquinone no addition), Item 2:control (menaquinone 100 μg/mL addition), Item 3: HYA 20 μM (menaquinoneno addition), Item 4: HYA 20 μM (menaquinone 100 μg/mL addition), Item5: linoleic acid 20 μM (menaquinone no addition), Item 6: linoleic acid20 μM (menaquinone 100 μg/mL addition). *, P<0.0001 vs. Control(ordinary one-way ANOVA)). HYA suppressed proliferation of Helicobacterpylori SS1 strain, TN2GF4 strain, TK1029 strain, RC-1 strain more thanlinoleic acid. Addition of menaquinone (MK-4) mitigated inhibition ofproliferation of Helicobacter pylori by fatty acid.

Example 4 Helicobacter pylori SS1 Strain and TN2GF4 Strain InhibitoryEffect (In Vivo) of Fatty Acid

A 5-week-old C57BL/6 female mouse was orally infected 3 times withHelicobacter pylori SS1 strain or TN2GF4 strain (1-5×10⁸ CFU) everyother day and dissected at 2 weeks from the final infection day, and thenumber of bacteria in the gastric mucosa was counted. The measurementand the number of bacteria in the gastric mucosa was performed asfollows. First, the greater curvature of the stomach of the mouse wasincised with scissors for ophthalmology, and the contents were washedaway with phosphate buffered saline (pH 7.4, PBS). Next, the mucousmembrane was scraped with a slide glass, 1 mL of PBS was added, themixture was sandwiched between ground glass parts of two glass slidesand evenly crushed to prepare a gastric mucosa suspension. The culturemedium was applied on a Helicobacter agar plate, cultured for 3 days andthe resulting colonies were counted. Water added with fatty acid (200μM) (HYA, HYB, KetoA, KetoC, linoleic acid, oleic acid, DHA, EPA) wasgiven to the mice from 1 week before infection. The control was free offatty acid addition (FIG. 3-1 A: infected with SS1 strain, FIG. 3-2 B:infected with TN2GF4 strain). HYA significantly suppressed the number ofHelicobacter pylori in both SS1 strain and TN2GF4 strain, as compared tothe control, and showed a strong suppressive action as compared to HYB,KetoA, KetoC, linoleic acid, oleic acid, DHA and EPA.

Example 5 Proliferation Inhibitory Effect (In Vivo) of HYA onHelicobacter Bacteria

A 5-week-old C57BL/6 female mouse was orally infected 3 times withHelicobacter pylori SS1 strain or TN2GF4 strain (1-5×10⁸ CFU) everyother day and dissected at 2 weeks from the final infection day, and thenumber of bacteria in the gastric mucosa was counted. Water added withHYA or linoleic acid (200 μM) was given to the mice from 1 week beforeinfection (FIG. 4-1 A: SS1 strain, FIG. 4-2 B: TN2GF4 strain). A5-week-old C57BL/6 female mouse was orally infected only once with astomach suspension of Helicobacter suis TKY strain or SNTW101strain-infected mouse and dissected 2 weeks later, and the number ofbacteria in the gastric mucosa was counted. Water added with HYA (200μM) was given to the mouse from one week before the infection (FIG. 4-3C). As a result, HYA inhibited proliferation of the Helicobacter pyloriSS1 strain, Helicobacter pylori TN2GF4 strain, Helicobacter suis TKYstrain, Helicobacter suis SNTW101 strain. The measurement of the numberof Helicobacter suis bacteria in the stomach of the infected mouse wasperformed as follows.

DNA was prepared from a part of the stomach tissue by using DNeasy Blood& Tissue Kit (Qiagen) and real-time PCR (model: CFX96 (Bio-Rad)) wasperformed. As the primer for Helicobacter suis quantification, thefollowing was used by reference to Diagn. Microbiol. Infect. Dis.46(1):1-7, 2003.

HeilF (SEQ ID NO: 1) (5′ AAG TCG AAC GAT GAA GCC TA 3′) HeilR(SEQ ID NO: 2) (5′ ATT TGG TAT TAA TCA CCA TTT C 3′)

The following were used as the primers for β-actin quantification of themouse by reference to J. Clin. Microbiol. 37:1958-1963, 1999.

(SEQ ID NO: 3) 5′ TCACCCACACTGTGCCCATCTACGA 3′ (SEQ ID NO: 4) 5′GGATGCCACAGGATTCCATACCCA 3′

For quantification by real-time PCR, iQTM SYBR Green Supermix was used.The reaction conditions were as follows.

1 cycle

95° C. 2 min

40 cycles

95° C. 5 sec

55° C. 15 sec

72° C. 45 sec

Thereafter, the temperature was increased by 0.5° C. every seconds from65° C. to 95° C. and the fluorescence was measured. Relativequantification was performed by multiplex reactions (same tube) andcomparative ΔΔCT method (ABI Prism 7700) by converting to numericalvalues by the heilmannii-suis gene quantitative ratio per β-actin geneamount and further adjusting to make the mean of the untreated to 1.

Example 6 Pathology Onset Suppressive Effect (In Vivo) of HYA on GastricMALT Lymphoma

Helicobacter suis is suspected to be the cause of the onset of gastricMALT lymphoma. A 5-week-old C57BL/6 female mouse was orally infectedonly once with a stomach suspension of Helicobacter suis TKYstrain-infected mouse and dissected 6 months after the infection, andthe number of bacteria in the gastric mucosa was counted according tothe method described in Example 5. Water added with HYA (200 μM) wasgiven to the mouse from one week before the infection.

(1) Measurement of the Number of Helicobacter suis Bacteria in theStomach of Infected Mouse

The measurement of the number of Helicobacter suis bacteria in thestomach of the infected mouse was performed in the same manner as inExample 5. The relative value of the number of bacteria in gastricmucosa when that with HYA non-administration is 1 is shown (FIG. 5-1 A,Item 1: HYA non-administration group, Item 2: HYA administration group).The number of bacteria was significantly suppressed in the HYAadministration group compared to the non-administration group.

(2) Histochemical Analysis

Ki-67 is known as a marker for cell proliferation and cell cycle.Proliferated cells in a tumor tissue were detected by immunostainingwith anti-Ki-67 antibody. A part of the stomach was fixed with 10%neutral formalin buffer to prepare a paraffin block, serial sectionswere cut out using a microtome and Hematoxylin Eosin (HE) staining wasperformed. Ki-67 (Clone SP6) rabbit monoclonal antibody (Thermo FisherScientific Inc.) diluted 1:300 was used as a dilution antibody,Histofine Simple Stain mouse MAX-PO® (NICHIREI BIOSCIENCES INC.,Code:414341) DAB staining (DAKO Inc.) was used as a secondary antibody,and Hematoxylin was used for comparison staining. As the negativecontrol, rabbit IgG antibody (Code No. X 0936 Lot 050 (DAKO Inc.)) wasused. The number of Ki-67 positive cells in lymphoid follicle when thatwith HYA non-administration is 1 is shown (FIG. 5-2 B, Item 1: HYAnon-administration group, Item 2: HYA administration group). An increasein the number of Ki-67 positive cells was significantly suppressed inthe HYA administration group compared to the non-administration group.

(3) Quantification of CD19, CD20 Expressions

CD19 and CD20 are cell surface markers for B-cell lymphoma. Usingreal-time reverse transcription PCR (real-time RT-PCR), the expressionlevels of CD19 and CD20 were measured. Using NucleoSpin (registeredtrade mark) RNA Kit (Takara Bio Inc.), RNA was prepared from mousegastric mucosa. Using PrimeScript™ RT Reagent Kit (Takara Bio Inc.),cDNA was prepared from RNA. For real-time RT-PCR, KAPA SYBR Fast ROX LowqPCR kit (KAPA BIOSYSTEMS Inc.) and QuantStudio7 Flex Real-time PCRSystem (Thermo Fisher Scientific Inc.) were used.

The following were used as the primers for quantification of CD19expression.

Fw: (SEQ ID NO: 5) 5′-AGTGACTAGCCTGGACTT-3′ Rv: (SEQ ID NO: 6)5′-ACTGACTGACACCATCTG-3′

The following were used as the primers for quantification of CD20expression.

Fw: (SEQ ID NO: 7) 5′-CAGGAAGAGTTTGGTCAA-3′ Rv: (SEQ ID NO: 8)5′-GGTTCACAGTCGTAGATAT-3′

The following were used as the primers for quantification ofglyceraldehyde-3-phosphate (GAPDH) expression.

Fw: (SEQ ID NO: 9) 5′-TGTGTCCGTCGTGGATCTGA-3′ Rv: (SEQ ID NO: 10)5′-TTGCTGTTGAAGTCGCAGGAG-3′

The conditions of 2-step real-time RT-PCR were as follows.

1 cycle

95° C. 3 min

40 cycles

95° C. 3 sec

60° C. 20 sec

The expression level of CD19 or CD20 relative to that ofglyceraldehyde-3-phosphate dehydrogenase (GAPDH) was converted to anumerical value and the value was further adjusted to make the averageexpression of CD19 or CD20 of the non-infection group 1. The CD20relative expression levels in the HYA administration group andnon-administration group are shown (FIG. 5-3 C, Item 1: non-infectiongroup, Item 2: HYA non-administration group, Item 3: HYA administrationgroup). Similarly, the CD19 relative expression levels in the HYAadministration group and non-administration group are shown (FIG. 5-4 D,Item 1: non-infection group, Item 2: HYA non-administration group, Item3: HYA administration group). As a result, the relative expression levelwas significantly suppressed in the HYA administration group compared tothe non-administration group.

Example 7 Proliferation Inhibitory Effect (In Vitro) of HYA onCampylobacter jejuni ATCC33560 Strain and Campylobacter coli ATCC33559Strain

Campylobacter jejuni ATCC33560 strain or Campylobacter coli ATCC33559strain preserved at −80° C. was applied on a CODA agar medium added withSR0155E and cultured for 2 days in an incubator (5% O₂, 10% CO₂, 85% N₂,humidity 100%, 42° C.). The resulting colonies were picked up,inoculated to 10% FBS-added Brucella liquid medium and shaken in theincubator overnight. 2 mL of 5% FBS-added Brucella liquid mediumcontaining Campylobacter jejuni ATCC33560 strain or Campylobacter coliATCC33559 strain at a concentration of 1×10⁶ CFU/mL was dispensed toeach well of a 12-well plate, fatty acid was added at an optionalconcentration (0, 100, 200, 400 μM), and the mixture was shaken in theincubator for 24 hr. After 24 hr, the absorbance (600 nm) of the liquidmedium after shaking was measured. On the other hand, the liquid mediumat 24 hr after shaking was diluted 1×10⁶-fold with BSG (PBS containing0.01% gelatin, pH 7.4), 0.1 mL was applied on 10% FBS-added Brucellaagar medium, cultured in the incubator for 2 days and the number of theresulting colonies was measured (FIGS. 6-1 to 6-4, C: control, LA 100:linoleic acid 100 μM, LA 200: linoleic acid 200 μM, LA 400: linoleicacid 400 μM, HYA 100: HYA 100 μM, HYA 200: HYA 200 μM, HYA 400: HYA 400μM, αHYA 100: αHYA 100 μM, αHYA 200: αHYA 200 μM, αHYA 400: αHYA 400 μM,γHYA 100: γHYA 100 μM, γHYA 200: γHYA 200 μM, γHYA 400:γHYA 400 μM). Asa result, HYA, αHYA, γHYA inhibited proliferation of Campylobacterjejuni ATCC33560 strain or Campylobacter coli ATCC33559 strain in aconcentration dependent manner, and showed a strong inhibitory actioncompared to linoleic acid.

From the above results, it was shown that a fatty acid having 18 carbonatoms and a hydroxyl group at the 10-position has an inhibitory actionon the proliferation of a bacterium provided with a menaquinonesynthesis route via futalosine or a futalosine derivative such asHelicobacter pylori, Helicobacter suis and the like, a suppressiveaction on an increase in the number of Ki-67 positive cells, asuppressive action on an increase in the CD19 expression level and CD20expression level, and a suppressive action on the onset of pathology ofgastric MALT lymphoma. In addition, it was shown that a fatty acidhaving 18 carbon atoms and a hydroxyl group at the 10-position has aninhibitory action on proliferation of Campylobacter jejuni andCampylobacter coli.

INDUSTRIAL APPLICABILITY

In the present invention, it was found that a fatty acid having 18carbon atoms and a hydroxyl group at the 10-position inhibitsproliferation of a bacterium provided with a menaquinone synthesis routevia futalosine or a futalosine derivative. The composition containingthe fatty acid can be used in various fields such as pharmaceuticalproduct, food, feed and the like, and is industrially extremely useful.

This application is based on a patent application No. 2017-053056 filedin Japan (filing date: Mar. 17, 2017), the contents of which areincorporated in full herein.

The invention claimed is:
 1. A method for inhibiting proliferation of abacterium provided with a menaquinone synthesis route via futalosine ora futalosine derivative, comprising administering a fatty acid having 18carbon atoms and a hydroxyl group at the 10-position to a subject. 2.The method according to claim 1, wherein the fatty acid has a cis doublebond at at least the 12-position.
 3. The method according to claim 2,wherein the fatty acid is at least one selected from the groupconsisting of 10-hydroxy-cis-12-octadecenoic acid,10-hydroxy-cis-12,cis-15-octadecadienoic acid and10-hydroxy-cis-6,cis-12-octadecadienoic acid.
 4. The method according toclaim 3, wherein the fatty acid is 10-hydroxy-cis-12-octadecenoic acid.5. The method according to claim 1, wherein the bacterium isHelicobacter bacterium.
 6. The method according to claim 5, wherein theHelicobacter bacterium is selected from the group consisting ofHelicobacter pylori and Helicobacter suis.
 7. The method according toclaim 1, wherein the bacterium is Campylobacter bacterium.
 8. The methodaccording to claim 7, wherein the Campylobacter bacterium is selectedfrom the group consisting of Campylobacter jejuni and Campylobactercoli.